Not applicable.
The invention relates generally to the fields of biochemistry, enzymology, and marine biology. More particularly, the invention relates to a purified enzyme useful for producing pseudopterosins.
Pseudopterogorgia elisabethae, a purple frilly seafan, is a gorgonian commonly found in the shallow-water reefs of the tropical Atlantic including regions of the Caribbean. P. elisabethae is of particular commercial importance as it has been found to contain numerous biologically active small molecule compounds. Among these, pseudopterosins (also known as terpenes, e.g., diterpenes) have been shown to exhibit anti-inflammatory and analgesic properties, and are currently being used as topical agents in skin care products. In the biosynthetic pathway shown in FIG. 1, pseudopterosin/seco-pseudopterosins are generated from geranyl geranyl diphosphate (GGPP). A key step in this pathway is the cyclization of GGPP to elisabethatriene (compound 18). From elisabethatriene, the intermediate compounds 19-27 and pseudopterosin A are made. The identification of enzymes responsible for catalyzing key steps in this pathway, however, has been elusive. Identification of such an enzyme would facilitate the development of a chemoenzymatic method for the production of marine diterpenes.
The invention relates to the purification and partial sequencing of an elisabethatriene cyclase from P. elisabethae. This cyclase is useful for converting GGPP to elisabethatriene, a step involved in the production of seco-psuedopterosins, pseudotopterosins, and related molecules. The purified enzyme is useful for synthesizing the foregoing molecules. In addition, the purified cyclase should be useful for making other diterpenes. The methods disclosed herein might also be used to produce eleutherobin, an antimitotic agent isolated from the soft coral Erythropodium caribaeorum. 
Accordingly, the invention features a purified elisabethatriene cyclase such as a purified protein isolatable from a Pseudopterogorgia elisabethae coral sample having an apparent molecular weight of about 47,000 Da; an isoelectric point of about 5.1; and the ability to cyclize geranyl geranyl diphosphate. The purified protein can be one that includes the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and/or SEQ ID NO:4. Fragments of the foregoing that are capable of catalyzing the formation of elisabethatriene from geranyl geranyl diphosphate are also featured in the invention.
In another aspect the invention features a method of purifying an elisabethatriene cyclase from a Pseudopterogorgia elisabethae sample. This method includes the steps of: (A) preparing a cell free extract from a Pseudopterogorgia elisabethae sample; (B) separating the cell free extract into at least two fractions, one that exhibits elisabethatriene cyclase activity and one that does not; and (C) collecting the fraction that exhibits elisabethatriene cyclase activity. The step (A) of preparing a cell free extract from the Pseudopterogorgia elisabethae sample can be performed by flash freezing the Pseudopterogorgia elisabethae sample using liquid nitrogen; homogenizing the frozen sample with a buffer and liquid nitrogen; separating the homogenized sample into a cellular portion and a non-cellular portion; and collecting the non-cellular portion. The step (B) of separating the cell free extract can be performed by subjecting the cell free extract to one or more chromatographic separation steps such as DEAE ion exchange chromatography, phenyl sepharose chromatography, hydroxyapatite chromatography, and/or ion exchange chromatography with 2-Propen-1-aminium, N,N,-dimethyl-N-2-propenyl-, chloride, polymer with 1,4-bis(1-oxo-2-propenyl) piperazine and 2-methyl-2-propenamide.
The invention also provides a method for cyclizing geranyl geranyl diphosphate, e.g., to make elisabethatriene. This method is performed by contacting geranyl geranyl diphosphate with a purified elisabethatriene cyclase under reaction conditions that result in the production of elisabethatriene. The elisabethatriene thus formed can be used as a substrate to produce other molecules involved in pseudopterosin synthesis. For example, elisabethatriene can be reacted to produce elisabethadione which can be reacted to produce elisabethadiol. The latter can be reacted to produce pseudopterosin agylcone which can be reacted to produce pseudopterosin A.
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Definitions of molecular biology terms can be found, for example, in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994. Definitions of organic chemistry and enzymology can be found, for example, in R. B. Silverman et al., The Organic Chemistry of Enzyme-Catalyzed Reactions, Academic Press: San Diego, Calif., 2000; and R. T. Morrisson et al., Organic Chemistry, 6th edition, Addison-Wesley Publishing Co.: Boston, Mass., 1992.
As used herein, the terms xe2x80x9cproteinxe2x80x9d and xe2x80x9cpolypeptidexe2x80x9d are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation. A xe2x80x9cpurifiedxe2x80x9d polypeptide is one that has been substantially separated or isolated away from other polypeptides in a cell, organism, or mixture in which the polypeptide occurs (e.g., 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of contaminants).
A xe2x80x9cpurified elisabethatriene cyclasexe2x80x9d is a purified protein isolatable from P. elisabethae that has the ability to cyclize GGPP. The phrase includes the purified native form of elisabethatriene cyclase isolatable from P. elisabethae and having an apparent molecular weight of about 47,000 Da and an isoelectric point of about 5.1. It also includes naturally occurring and non-naturally occurring proteins having a similar structure (e.g., sharing 65, 70, 75, 80, 85, 90, 95, 97, 98, 99% or more sequence identity) and enzymatic activity, e.g., allelic variants of a native elisabethatriene cyclase, mutants of a native elisabethatriene cyclase, and forms of the enzyme produced by recombinant DNA technology or chemical synthesis.
A xe2x80x9cfragmentxe2x80x9d of an elisabethatriene cyclase polypeptide is a portion of an elisabethatriene cyclase polypeptide that is less than full-length (e.g., a polypeptide consisting of 5, 10, 15, 20, 30, 40, 50, 75, 100 or more amino acids of native elisabethatriene cyclase polypeptide), and preferably retains at least one functional activity of native elisabethatriene cyclase polypeptide (e.g., the ability to cyclize a GGPP substrate).
The term xe2x80x9cantibodyxe2x80x9d includes polyclonal and monoclonal antibodies as well as antibody fragments or portions of immunolglobulin molecules that can specifically bind the same antigen as the intact antibody molecule.
As used herein, xe2x80x9cbind,xe2x80x9d xe2x80x9cbinds,xe2x80x9d or xe2x80x9cinteracts withxe2x80x9d means that one molecule recognizes and adheres to a particular second molecule in a sample, but does not substantially recognize or adhere to other structurally unrelated molecules in the sample. Generally, a first molecule that xe2x80x9cspecifically bindsxe2x80x9d a second molecule has a binding affinity greater than about 105 to 106 liters/mole for that second molecule.
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions will control. In addition, the particular embodiments discussed below are illustrative only and not intended to be limiting.